In life sciences and in related applied fields there is a need for non-antibody polypeptide molecules capable of performing specific protein-protein interactions. A main focus relies on identifying polypeptide domains that bind to a predetermined target. However, a major obstacle of linear polypeptides comprising about 5 to about 50 amino acids is their intrinsic flexibility. In solution such polypeptides are usually transitioning a large number of structural states that are almost equivalent from an energetic perspective. Nevertheless, such structural states are generally highly dependent on the environment of the polypeptides. As the structural state is an important factor for presenting a certain epitope, e.g. if such polypeptides are used for immunization of an animal for antibody production, it is an essential requirement that the structural state of the polypeptide is not affected by environmental changes, such that an unambiguous presentation of a certain structural state representing a defined epitope can be ensured.
To meet those demands, a protein scaffold can be used where the polypeptide of interest is grafted into a rigid structure. The scaffold forces the polypeptide insertion into an entropy-restricted, structural state, limiting its torsional degrees of freedom. These constructs can be used for applications, such as for the immunization of an experimental animal for producing antibodies against the polypeptide insertion. Furthermore, such a scaffold can be used for the purpose to map antibody epitopes. In another application, such a scaffold can be used as a chimeric calibrator polypeptide for diverse immunological assays. In another application such a scaffold can display constrained peptides with predefined target binding specificity, which allows the scaffold to be used in diverse affinity purification approaches, like affinity chromatography or pull-down assays. In another application, antibody CDR loops can be grafted into such a scaffold. In another application, subdomains of other proteins can be grafted into such a scaffold in order to circularly permutate the chimeric target polypeptide. Domains such as variable loops of antigen binding regions of antibodies have been extensively engineered to produce amino acid sequence segments having improved binding (e.g. affinity and/or specificity) to known targets (e.g. disclosed in Knappik, A. & Plückthun A. J. Mol. Biol. 296 (2000) 57-86; EP 1025218). Engineering of non-antibody frameworks has been reviewed e.g. by Hosse, R. J. et al. Protein Sci., 15 (2006) 14-27. Non-antibody or alternative protein scaffolds have considerable advantages over traditional antibodies due to their small size, high stability, and ability to be expressed in prokaryotic hosts. Novel methods of purification are readily applied; they are easily conjugated to drugs/toxins, penetrate efficiently into tissues and are readily formatted into mono- or multi-specific binders (Skerra, A, et al. J. Mol. Recognit. 13 (2000) 409-410; Binz, H. K. et al. Nature Biotechnol. 23 (2005) 1257-1268).
As known in the art, human FKBP12 can be used as a protein scaffold to improve its enzymatic activity. Knappe, T. A., et al. (J. Mol. Biol. 368 (2007) 1458-1468) reported that the Flap-region of human FKBP12 can be replaced by the IF domain of the structurally related E. coli chaperone SlyD. This chimeric FKBP12-IF polypeptide Thermus thermophiles SlyD-FKBP has a 200 times increased peptidyl-prolyl-cis/trans isomerase activity (PPI activity) compared to the isolated polypeptide. The E. coli SlyD and human FKBP12 (wild type and mutants C23A and C23S) can be recombinantly produced in E. coli in high yield in soluble form (Standaert, R. F., et al., Nature 346 (1990) 671-674).
SlyD derived from thermophilic organisms and E. coli SlyD can be used as chaperones in the recombinant expression of chimeric polypeptides in E. coli (Ideno, A., et al., Appl. Microbiol. Biotechnol. 64 (2004) 99-105). The E. coli SlyD and FKBP12 polypeptides are reversibly folding polypeptides (Scholz, C., et al., J. Biol. Chem. 271 (1996) 12703-12707).
The amino acid sequence of the human FKBP12 polypeptide comprises a single tryptophan residue at position 60. Thus, human FKBP12 mutants can be analyzed for structural integrity simply by analyzing the tryptophan fluorescence (DeCenzo, M. T., et al., Protein Eng. 9 (1996) 173-180). A test for remaining catalytic activity of the human FKBP12 mutant can be performed by determining the remaining rotamase activity (Brecht, S., et al., Neuroscience 120 (2003) 1037-1048; Schories, B., et al., J. Pept. Sci. 13 (2007) 475-480; Timerman, A. P., et al., J. Biol. Chem. 270 (1995) 2451-2459). It is also possible to determine the structural integrity of human FKBP12 mutants by determining the FK506- or Rapamycin binding (DeCenzo, M. T., et al., Protein Eng. 9 (1996) 173-180). McNamara, A., et al. (J. Org. Chem. 66 (2001) 4585-4594) report peptides constrained by an aliphatic linkage between two C (alpha) sites: design, synthesis, and unexpected conformational properties of an i,(i+4)-linked peptide.
Suzuki, et al. (Suzuki, R., et al., J. Mol. Biol. 328 (2003) 1149-1160) report the three-dimensional solution structure of an archaic SlyD with a dual function of peptidyl-prolyl-cis-trans isomerase and chaperone-like activities. Expression vector, host, fused polypeptide, process for producing fused polypeptide and process for producing protein are reported in EP 1 516 928. Knappe, T. A., et al., reports that the insertion of a chaperone domain converts human FKBP12 into a powerful catalyst of protein folding (J. Mol. Biol. 368 (2007) 1458-1468). A chimeric polypeptide with superior chaperone and folding activities is reported in WO 2007/077008. In WO 03/000878 the use of SlyD chaperones as expression tool is reported. In EP 1 621 555 an immunogen, composition for immunological use, and method of producing antibody using the same are reported. Rebuzzini, G. (PhD work at the University of Milano-Bicocca (Italy) (2009)) reports a study of the hepatitis C virus NS3 helicase domain for application in a chemiluminescent immunoassay.
In WO 2007/077008 chimeric fusion proteins with superior chaperone and folding activities are reported. The conversion of human FKBP12 into a powerful catalyst of protein folding by insertion of a chaperone domain is reported by Knappe et al. (Knappe, T. A., et al., J. Mol. Biol. 368 (2007) 1458-1468).
WO 2012/150320 discloses a fusion polypeptide comprising one or more fragments of one or more peptidyl-prolyl cis/trans isomerase or FKBP domain family members and its use in methods for antibody screening/selection, for epitope mapping as well as its use as immunogen for the production of antibodies specifically binding an immunogenic peptide or secondary structure presented by the fusion polypeptide.
Among other SlyD chaperones from different species, especially suited is the Thermus thermophilus SlyD FKBP domain (herein also referred to as TtSlyD-FKBP) due to its superior biophysical properties regarding thermodynamic stability and solubility (Low et al. (2010) J Mol Biol 398(3): 375-390). The Thermus thermophilus SlyD FKBP domain can be used as scaffold for the presentation of constrained peptides WO 2012/150320 which is useful for various applications, such as display methods including phage display, ribosome display, mRNA display and cell surface display. Such methods can be applied to select and optimize target-binding polypeptides from libraries with a large number of candidate amino acid sequences. Another application of the Thermus thermophilus SlyD FKBP domain with a certain constrained peptide bound thereto is its use as immunogen for the production of antibodies in animals (WO 2012/150320). Further, the Thermus thermophilus SlyD FKBP domain with a certain constrained peptide can be used as a ligand in protein-protein interaction experiments, whereas the constrained peptide represents one specific binding site of the corresponding entire protein binding partner.
These methods and experiments require as a tool an antibody which specifically binds to the Thermus thermophilus SlyD FKBP domain (herein referred to as anti-TtSlyD-FKBP antibody). No such antibody is described in the state of the art. The problem to be solved by the present description is therefore the provision of an antibody which binds to the native TtSlyD-FKBP polypeptide.